Our long-term goal is to develop a complete understanding of the spatial and temporal events involved in the regulation of RNA polymerase II transcription and pre-mRNA processing in living cells. By understanding these parameters in the context of the living cell, we will have the basis to identify changes in these events that occur in cells or tissues associated with various disease states. Our short-term goals are to elucidate the dynamics of gene expression in real-time at the single cell level. We have developed a live cell system that allows us to directly visualize an inducible genetic locus and its RNA and protein products in living cells. Using this system we will: determine the order of recruitment of the gene expression machinery to a regulatable genetic locus in living cells, determine the turnover of proteins at the transcription site, examine the dynamics of chromatin remodeling upon transcriptional down-regulation, and determine how transcription and DNA replication are spatially coordinated at a specific DNA locus. We will also determine what regulates the organization of the gene expression machinery during mitosis/G1. Finally, we will characterize a novel protein, Btf, that may be involved in coordinating transcription and pre-mRNA splicing. It is important to understand the functional organization of the nucleus as aberrations in nuclear organization have the potential to result in alterations in gene expression, the end product of which may be one of the many pathologies associated with human disease.